Post translational modifications of proteins through phosphorylation play a role in many cellular processes such as the transduction of extracellular signals, intracellular transport, and cell cycle progression. Our group is interested in the study of signaling events that are involved in sperm differentiation and function. Although meiotic events are well characterized in the female germ cells, the regulation of male germ cell meiosis and differentiation into mature sperm is still not well understood. Recently, a novel family of serine/threonine kinases (testes specific serine kinases (Tssk)) has been found post meiotically expressed in the male germ cells. We have demonstrated the presence of 1or more members of the Tssk family in mature mammalian sperm (Hao et al., 2004) (and PR) and in addition, we have obtained evidence that Tssk homologues are expressed in the testes of invertebrates. The conserved testicular expression pattern of Tssk genes, as well as the importance of phosphorylation in signaling processes, strongly suggests that Tssk(s) have important role(s) in germ cell differentiation and/or sperm function. This proposal is designed to set the grounds to understand the function(s) of Tssk(s) in germ cells and in mature sperm. Future directions of this project include the analysis of null mutants (KO) of Tssk(s), as well as the investigation of signaling pathways involving Tssk(s) using genetic methods available in invertebrate models. Toward these goals, it will be necessary first to create tools for these studies and to characterize these kinases. Taking this into consideration, this application will focus on: 1) The generation of specific antibodies against the 5 members of the Tssk kinase family; 2) The identification of the Tssk(s) present in mature sperm and the investigation of their subcellular localization; 3) The biochemical characterization of Tssk4 kinase activity and the study of potential interacting partners. Although not part of this application, in collaboration with Dr. Mitch Eddy from NTH, a mouse KO of Tssk4 is in the process of being produced. Importantly, the proposed research will be useful to begin to illuminate the pathways of Tssk gene function. The predicted importance of Tssks in spermiogenesis and/or sperm function has been recently confirmed by phenotypic analysis of Tssk6 KOs (also known as Sstk)(Spiridonov et al., 2005); these null mutant mice present a sterile phenotype in the males but not in the females. The tools generated as part of this application will be very important for the analysis of Tssk KO phenotypes and to start the study of Tssk function. The predicted importance of Tssks in spermiogenesis and/or sperm function suggests that this research can ultimately help to design novel human contraceptives. [unreadable] [unreadable] [unreadable]